Species of Scenedesmus and Desmodesmus are ubiquitous in freshwater ecosystems. However, phenotypic plasticity coupled with the minute variations between named species make the identification of taxa by light microscopy extremely difficult, especially from field samples. As a result, the actual level of diversity and distributions of these genera are not known. In this study, the diversity of Scenedesmus and Desmodesmus from Itasca State Park (ISP), Minnesota, USA was studied using culture techniques followed by light microscopy and internal transcribed spacer (ITS-2) rDNA sequence analysis. From 100 characterized isolates of Scenedesmus, Desmodesmus and allied isolates, 34 different sequence types were detected, 24 of which are different enough from each other to correspond to distinct taxa. This diversity greatly exceeds that described for these genera in previous studies of ISP algae using light microscopy alone. The sequences of five ISP isolates identically matched those of previously described species or isolates, and sequences of six ISP isolates differed by eight bases or less from those of previously described species. The isolation of Desmodesmus species from Itasca State Park with sequences identical to those from isolates from highly disjunct locations around the world suggests that these species are widely distributed. Results from this study also indicate that isolates with very similar (< 8 substitutions) ITS-2 sequences tend to have only very slight morphological variation. We conclude that culturing and ITS-2 sequence analysis is an effective technique for investigating species level diversity in Scenedesmus and Desmodesmus.